Often, high-sensitivity, point-of-care (POC) clinical tests, such as HIV viral load, require large volumes of plasma. Although centrifuges are ubiquitously used in clinical laboratories to separate plasma from whole blood, centrifugation is generally inappropriate for on-site testing. Suitable alternatives are not readily available to separate the relatively large volumes of plasma from milliliters of blood that may be needed to meet stringent limit-of-detection specifications for low-abundance target molecules. The present disclosure provides, inter alia, a simple-to-use, low-cost, pump-free, membrane-based, sedimentation-assisted plasma separator capable of separating a relatively large volume of plasma from undiluted whole blood within minutes. In one embodiment, this plasma separator includes of an asymmetric, porous, polysulfone membrane housed in a disposable chamber. The separation process takes advantage of both gravitational sedimentation of blood cells and size exclusion-based filtration. An exemplary plasma separator demonstrated a “blood in—plasma out” capability, consistently extracting 275±33.5 μL, of plasma from 1.8 mL of undiluted whole blood within less than 7 min. The device was used to separate plasma laden with HIV viruses from HIV virus-spiked whole blood with recovery efficiencies of 95.5% ±3.5%, 88.0% ±9.5%, and 81.5% ±12.1% for viral loads of 35 000, 3500, and 350 copies/mL, respectively. The separation process is self-terminating to prevent excessive hemolysis. The HIV-laden plasma was then injected into a exemplary microfluidic chip for nucleic acid testing and was successfully subjected to reverse-transcriptase loop-mediated isothermal amplification (RTLAMP), demonstrating that the plasma is sufficiently pure to support high-efficiency nucleic acid amplification.
Over two-thirds of the estimated 34 million people living with HIV/AIDS worldwide reside in developing countries, and nearly three-fourths of the 2.5 million new HIV infections in 2011 occurred in these countries. HIV viral load testing plays a critical role in clinical decisions on when and whether to switch to second-line treatment; in optimizing the duration of first-line treatment by detecting occult nonadherence; in diagnosing HIV infection in babies under 18 months of age, born to HIV-infected mothers, in whom the presence of HIV antibodies is not indicative of the disease; and in detecting early newly infected individuals during the seroconversion window period when antibodies are present at undetectable concentrations. Although a standard practice in developed countries, HIV viral load determination is not widely used in low- and middle-income countries, because of technical constraints, lack of testing facilities, lack of trained personnel, and cost. There is an urgent need to develop an affordable, simple, easy-to-use point-of-care (POC) diagnosis technology for HIV viral load testing in resource-constrained settings.
Usually, plasma separation from raw whole blood is required for HIV viral load testing, as the presence of blood cells and components in the sample, such as hemoglobin and lactoferrin, may inhibit DNA polymerase and lead to low amplification efficiency, inaccurate quantification, and even amplification failure. In addition, prevailing HIV viral load standards are based on the number of virus copies in a unit volume of plasma—not whole blood. In clinical laboratories, plasma separation is typically carried out with a benchtop centrifuge. Separation of relatively large volumes of plasma from whole blood remains a challenge in resource-constrained settings, because of the lack of laboratory infrastructure.
Various microfluidic approaches have been developed to separate plasma from whole blood at the point of care. Such approaches, however, work with small (<100 microliter) volumes of blood and plasma that are insufficient for conventional nucleic acid-based molecular diagnostics such as PCR.
To overcome the shortcomings of the above devices, some have used a centrifugation approach that requires a high-speed spinner and electrical power, which may not be readily available in resource limited settings. Others have used filtration-based separation, but existing separation-based methods require extensive dilution that adversely affects the limit-of-detection, and the limit-of-detection is critical in viral load detection.